This document discusses production of disease-free seedlings through tissue culture techniques. It begins with an introduction to meristem culture and how it can be used to eliminate viruses from plant materials. It then provides the step-by-step protocol for conducting meristem culture, including explant selection, surface sterilization, culture establishment, and acclimatization of plantlets. Applications of meristem culture like virus elimination, micropropagation, and facilitating international exchange of germplasm are also summarized. The document lists some advantages and disadvantages of the technique.

1. Presented by:
Suresh Yadav
I.D No.- Palm 6005

3. Production of disease free seedlings
through tissue culture
Presented by :- Suresh Yadav
I.D No:- PALM 6005

4. Tissue culture :-Morel and Martin (1952) developed the
technique of meristem culture for in vivo virus eradication of
Dahlia.
Shoots of all angiosperms and gymnosperms grow by virtue of
their apical meristems.
The apical meristem is usually a dome of tissue located at the
extreme tip of a shoot and measures approx. 0.1 mm in diameter
and 0.25 to 0.3 mm in length.
Meristem or shoot tip is isolated from stem by applying a V-
shaped cut.
MS medium salts have been very satisfactory for such cultures
though White’s and Gautheret’s were the most widely used media
during the early days of meristem culture.
INTRODUCTION

7. PROTOCOL
Remove the young twigs from a healthy plant. Cut the tip (1 cm)
portion of the twig
Surface sterilize the shoot apices by incubation in a sodium hypochlorite
solution (1% available chlorine) for 10 minutes. The explants are
thoroughly rinsed 4 times in distilled water
Transfer each explants to a sterilized petri dish.

8. Remove the outer leaves from each shoot
After the removal of all outer leaves, the apex is exposed.
Cut off the ultimate apex with the help of scalpel and transfer only those less
than 1 mm in length
Incubate the culture under 16hrs light at 25°C
As soon as the growing single leafy shoot or multiple shoots obtained from
single shoot tip or meristem, transfer them to hormone free medium to develop
roots.
The plants are later transferred to pots containing compost and kept under
green house condition for hardening.

9. Application of Shoot-tip or Meristem
Culture
1. Virus Elimination
• Plants are often infected with more
than one type of virus, including
some even not known.
• A general term virus- free is used
by commercial horticulturist by
this method.

10. 2. Micro Propagation
• Asexual or vegetative propagation
(vegetative part) of whole plants
using tissue culture techniques
referred to as micro propagation.
3. Storage of Genetic Resources
• Many plants produce seeds that are
highly heterozygous in nature or that
is recalcitrant. Such seeds are not
accepted for storing genetic resources.
So , the meristem from such plants can
be stored in vitro.

11. 4. Use in Plant Breeding:
•In many plant breeding experiments the hybrid plants produce abortive
seeds or non viable seeds. As a result, it makes a barrier to crossibility in
plants where non-viable seeds are unable to develop into mature plants.
Shoot-tip or meristem from such hybrid plant can be cultured to speed up
breeding programme.
5. Quarantine
• Plantlets derived from shoot-tip or meristem
cultures are easily accepted by the quarantine
authority for international exchange without any
checking.
• Therefore, using this technique , crop plants can
be easily exchanged in crop improvement
programmes that are based on materials from
different parts of the world.

12. List of the plants from which viruses have been eliminated by
meristem cultures

13. ADVANTAGES:
Lack of vascular tissue.
High auxin concentration.
Production of virus free plants
Facilitation of exchange between locations
Cryopreservation or in-vitro conservation of germplasm
DISADVANTAGES:
Isolation is difficult
Low survival rate & regeneration time for explants may be long(about 8
months for potato explant)
Removal of explant causes a setback in the growth of mother
plant.

14. Seed Health Testing

15. Content
• Introduction
• Objectives
• Seed Health Testing for Fungi
• Detecting Seed Borne Bacterial Plant
Pathogens
• Detecting Seed Borne Plant Viruses
• Detection of Insects and Nematodes
• Conclusion
• References

16. Introduction
• Seed Health Testing is a Science of determining the
presence or absence of disease causing agents, such
as fungi, bacteria and viruses.
• Animal pests like eelworms, insects in the seed
samples.

17. Objectives
• To determine the health status of a seed lot which in
turn establishes the sanitary condition of the seed in
commerce.
• To obtain objective proof of whether the seed lot meets
the requisite certification standards or not.
• To obtain objective proof of whether the lot meets
requisite quarantine requirements.

18. Methods of seed Heath Testing for
Fungi
• Visual examination –
with or without
stereomicroscope.
• Inspected in dry state for
the presence of
impurities such as ergots
or other sclerotia.

19. Determination of Karnalbunt (Neovassia
indica) of wheat
• Infected seeds have a
characteristic black powdery
mass generally along with the
suture.
• Severe infection – most of the
endosperm together with
pericarp and aleurone layer gets
destroyed giving grains a boat
like appearance.

20. Determination of bunt (Neovossia
horrida) of Rice
•Seeds are opened with the
help of the blade
•Whole grains
transformed into black
powdery mass

21. • Alternate procedure : Paddy seeds are
soaked in 0.2% solution of sodium hydroxide
for 24 hours at 18 -25°C .
• Swollen seeds are visually examined for shiny
jet black colour.

22. Determination of Loose smut
infection in Wheat
• Soak seeds for 24hours at 20°C in Sodium Hydroxide and
Trypan Blue.
• Separate the embryos by passing the soaked material along
with warm water (50-60°C)
• Embryos are washed in wire basket and dehydrated in 95%
alcohol for 2min and later transferred to a mixture of
lactophenol and water.

23. Continue:-
• Further separation of embryos can be made.
• Embryos are placed in lactophenol and boiled.
• Embryos are examined in microscope.
• Infected fungus with loose smut fungus is present as hyphal
strands.
• Golden brown thread like mycelium which is septate with non
uniform thickness and swellings are classified as infected
embryos.

24. UV Examination
• Toxins in Fungi gives Fluoroscence
appearance.
• This represents presence of Fungi

25. Blotter Method
•Seeds are placed on moistened blotters, filter papers at least 20mm apart.
•Blotters are placed in closed containers and incubated for certain number
of days and later examined for pathogens

26. Agar Plate Method
•After pretreatment seeds are spaced on the surface of 2% malt
extract sterilised agar.
•After plating dishes are incubated at 20-25°C for 5-8 days.

27. Wash Method
• Seeds are immersed in water with a wetting agent and
shaked vigorously to remove fungal spores, hyphae
etc.
• Excess liquid is removed and material is examined.

28. Examination of Imbibed Seeds
• Seeds are immersed in water, or other liquid to
make fruiting bodies, symptoms more easily
viable or to encourage the liberation of spores.
• After imbibition the seeds are examined with a
stereoscopic microscope

29. PCR Method
• It is a kind of molecular method.
• Specific primers are used in this method.
• PCR is a promising tool for distinguishing
specific sequences from a complex mixture of
DNA and therefore is useful for determining
the presence and quantity of pathogen-
specific or other unique sequences within a
sample

30. Methods to detect seed borne
bacterial pathogens
• Various methods are been developed.
• Some of the methods are simple and some are
specialized namely serological methods.

31. Growing out test
 The ‘growing out’ bioassay of a working seed
sample involves the sowing of test seeds into
seedlings under conditions optimal for the
disease development in glass house or closed
environmental chambers.
 ‘Growing out’ test has been successfully used
for a large number of Xanthomonads and
pseudomonas.

32. Indicator test
Working seed sample is sterilized with (2.6%) sodium-
hypochlorite for 15 min, and rinsed with sterile water.
The seed sample is incubated for 18-24 h in sterile water.
The water suspension is inoculated by infiltration into the
primary leaf node of 10 day old bean seedlings.
The appearance of lesions followed systemic necrosis is
positive reaction.

33. Serological Technique
 Serological tests are based on ‘In vitro’ reactions
between antigens and antibodies.
 This specific recognition of antigens by antibody has
offered the basic principle for the development of
various serological methods for detection and
identification of phytobacteria.
 The washing of the working seed samples are cultured
for 36 hr using sterile distilled water.
 The supernatant is tested with antiserum of the
suspected pathogen.

34. Culture Method
• By culturing the samples
on the particular media.
• Presence of bacteria can
be observed on the media.

35. Methods to detect seed borne Plant
Viruses
Biological Method
a) By growing seeds: The seeds are examined and abnormal
seeds are separated.
b) Both normal looking and abnormal looking seeds are grown
separately and the seedings are observed for the characteristic
symptom of the virus disease.
c) Plants are kept free of insect vectors such as mites and white
fly.

36. • Direct seed Test
a) Seed are examined and abnormal seeds are
separated and handled separately.
b) Seeds are soaked in aqueous medium and then
triturated.
c) The slurries produced are then applied to
indicator or test plants.
d) The slurries containing virus produce symptoms
typical for a given virus on the indicator plant.

37. Serological Tests
• Double diffusion test
a) Seeds to be tested are soaked in tapwater.
b) Seed or part (embryo) of it is triturated and
triturate is transferred to a well cut in a
diffusion media(agar gel).
c) Antiserum specific for a suspect virus in a
seed is placed in separate well.

38. • In time the virus particles(antigen) and
antibody molecules diffuse towards one
another.
• Since diffusion is in two direction it is called
Double diffusion.

39. ELISA(Enzyme linked immunosobent
assay)
• In this procedure , antiserum
specific for a given virus is used
to coat the polystyrene plate.
• Antibody molecules become
absorbed.
• Then seed sample is added to the
plate.
• It is followed by adding enzyme
labelled specific antibody to the
plate.

40. • The enzyme alkaline phosphates is conjugated to the antibody
molecules specific for the virus under examination.
• Finally enzyme substrate is added to the plate.
• Hydrolyzed substrate is determined by measuring the
extinction spectrophotometrically or by visual observation.

41. Detection of Insects and Nematodes
Detection of presence of Insects
• Examined under magnification or stereoscopic microscope
• Result is mentioned as number of insects per weight of the
sample

42. Detection of external insect injury
• Working sample is Examined under magnification(10x) or
stereoscopic microscope.
• The absence of insects does not however guarantee that the
seed lot is free from insect infestation, i.e internal injury to the
seeds or internal infestation.

43. Detection of Nematode Infestation
• Working sample is visually examined for the presence of
ear cocke galls.
(hard, small, dark purplish- black colour structures)
• Galls are separated and soaked in water for 30 minutes.
and are cut in water in petridish for observing the release
of nematode larvae, galls releasing nematode are counted
and result is reported in percentage.
Ear cockle of wheat (Anguina tritici)

44. Galls on wheat
Anguina tritici

45. Root knot Nematode of Sweet Potato
(Meloidogyne incognita)
• Entire submitted sample is examined for
visible symptoms of nematode infestation.
Galls on Sweet Potato

46. • Nematode infested tubers look ugly and
disfigured.
• When tubers are cut across the knot , small
glistening round pin head shaped bodies are
seen and such tubers are separated and the
results are reported.

47. POTATO CERTIFIED SEED STANDARDS
Disease
Incidence
India Holland* Canada UK
Mild mosaics 3 2 1 5
SM and PLRV 1 0.25 1 2
Brown rot 0.014 0 0 0
LB, Dry rot 1 NP NP NP
Wet rot 0 NP NP NP
Black scurf 5** NP NP NP
Common scab 5** NP NP NP
Total tuber Disease 5 - - -
Root knot nematode Nil NP NP Nil
Cyst nematode Nil NP NP Nil
NP : No limit prescribed * : Brown rot prevalent since
1995
** : Tuber treatment compulsory

48. SEED CERTIFICATION STANDARDS FOR
FOUNDATION AND CERTIFIED SEEDS
Permissible DI limits for foliar diseases
Seed grade
% DI limits
Off typeMild
mosaic
Severe
mosaic/PLRV/PVT/ LST/
yellows
PSTV
FS-1 1.0 0.5
-
0.05
FS-II 2.0 0.75
-
0.05
Certified 3.0 1.0 - 0.1

49. PERMISSIBLE LIMITS FOR TUBER DISEASES
Grade
% incidence
Common
scab
Black
scurf
Cut/
bruised
Late
blight
Dry rot
FS-1 5.0 5.0 1.0 1.0 1.0
FS-II 5.0 5.0 1.0 1.0 1.0
Certified 5.0 5.0 5.0 1.0 1.0

50. Eligibility criteria for production of
minitubers through tissue culture
Basic Material
The basic tissue culture material must be of a notified variety and the plantlet should be tested by the accredited laboratory
for freedom from
 PVA, PVS, PVX, PVY, PVM, PLRV, PALCV, PSTVd, endophytic bacteria and fungi
 10 plantlet to be tested for each variety
 Method
 ELISA, RTPC
 Light microscopy
 Culturing on media

51. Laboratory & Green House
 Lab and net house facilities free from insect pests
 Growth and Potting medium – sterilized
 Net house facility
 Insect proof
 Double door with changing space

52.  All micro-propagation and green house
facilities must meet the prescribed standards
 Net house soil should be free from
 Wart
 Brown rot
 Non-cyst forming nematodes
 Cyst nematodes
 Common scab
CONTROLLED ENVIRONMENT
REQUIREMENT

53.  The seed producer should inform the competent authority well in advance
 Source of seed has to be tissue culture grown plantlets/ micro tubers
 A minimum of three inspections shall be made
 First inspection (35 days : plains) (45 days : hills)
- For diseases, off types & isolation (1 m)
 Second inspection
Besides general inspection pathogen testing (viruses) 5
(minimum) – 25 (maximum) plants/varieties
 Third inspection (immediately after haulm cutting)
- To check date of haulm cutting?
- Re-growth
INSPECTION OF NET HOUSE FACILITY

54. Use of effective sanitation
Sterilization of nursery beds
The net house grown crop should be 100%
free from all viruses
If any of the virus is detected, the crop will be
rejected
INSPECTION OF NET HOUSE FACILITY

55. Certification standards for net house
grown crop
Factor
Maximum
Permissible limits (%)
Off types 0.05
Mild mosaic 0.05
Severe mosaics, Apical
Leaf Curl, PLRV
0.05
Bacterial wilt Nil

56. Standards for mini tubers
Factor Standards
Weight (minimum) >1.0 g
Germination (minimum) 90%
Varietal purity
(minimum)
99%
Virus (all) 0.1